低聚壳聚糖硒抗玉米赤霉烯酮对IPEC-J2细胞紧密连接蛋白表达的影响

doi:10.16431/j.cnki.1671-7236.2021.11.041

摘要/Abstract

摘要： 试验旨在探究低聚壳聚糖硒抗玉米赤霉烯酮（zearalenon，ZEN）对猪小肠上皮细胞（IPEC-J2）紧密连接蛋白表达的影响。试验分为对照组（C）、ZEN （30 μg/mL ZEN）、ZEN+0.5 Se （30 μg/mL ZEN+0.5 μmol/L低聚壳聚糖硒，以Se计）、ZEN+1.5 Se和ZEN+3 Se组。待细胞长至60%~70%汇合时，ZEN+0.5 Se、ZEN+1.5 Se和ZEN+3 Se组培养液更换为含对应Se浓度的培养液，C和ZEN组更换为正常培养液，12 h后向含ZEN组加入30 μg/mL ZEN，继续培养24 h。收集各组细胞培养液，检测碱性磷酸酶（AKP）活性；Western blotting法检测闭锁连接蛋白（zonula occludens-1，ZO-1）、闭合蛋白（Occludin）的表达，免疫荧光法检测ZO-1和Occludin蛋白的表达和定位情况。AKP活性检测结果表明，与对照组相比，ZEN、ZEN+0.5 Se、ZEN+1.5 Se组AKP活性均显著升高（P<0.05），ZEN+3 Se组AKP活性差异不显著（P>0.05）；与ZEN组相比，ZEN+1.5 Se和ZEN+3 Se组AKP活性均显著降低（P<0.05）。Western blotting检测结果表明，与对照组比，ZEN组ZO-1和Occludin蛋白表达水平均显著降低（P<0.05）；与ZEN组比，ZEN+0.5 Se、ZEN+1.5 Se和ZEN+3 Se组ZO-1表达水平均差异不显著（P>0.05），但随着Se浓度的增加，ZO-1表达水平逐渐升高，ZEN+0.5 Se、ZEN+1.5 Se、ZEN+3 Se组Occludin表达水平均显著升高（P<0.05）。免疫荧光结果表明，低聚壳聚糖硒可缓解ZEN引起的ZO-1和Occludin蛋白荧光信号减弱现象，并恢复ZO-1和Occludin蛋白的定位分布。由此说明，低聚壳聚糖硒可降低ZEN引起的IPEC-J2细胞AKP活性升高，上调ZEN引起的ZO-1、Occludin蛋白表达水平下降，并调节其定位分布。

关键词: 玉米赤霉烯酮; 低聚壳聚糖硒; 猪小肠上皮细胞(IPEC-J2); 闭锁连接蛋白(ZO-1)

Abstract: The aim of this study was to investigate the effects of selenium-oligochitosan against zearalenon (ZEN) on the expression of tight junction protein of IPEC-J2 cells. The experiment was divided into control group (C), ZEN (30 μg/mL), ZEN+0.5 Se (30 μg/mL ZEN+0.5 μmol/L selenium-oligochitosan, in terms of Se), ZEN+1.5 Se and ZEN+3 Se groups. When the confluence of cells reached 60%-70%, the culture medium of ZEN+0.5 Se, ZEN+1.5 Se and ZEN+3 Se groups was replaced with the culture medium containing the corresponding Se concentration, C and ZEN groups were replaced with normal culture medium. After 12 h, 30 μg/mL ZEN was added to the ZEN-containing groups, and the culture was continued for 24 h. The cell culture medium of each group was collected to detect the activity of alkaline phosphatase (AKP), the expressions of zonula occludens-1(ZO-1) and Occludin were detected by Western blotting, the expression and localization of ZO-1 and Occludin proteins were detected by immunofluorescence. The results of AKP activity showed that compared with control group, the AKP activity was significantly increased in ZEN, ZEN+0.5 Se and ZEN+1.5 Se groups (P<0.05), but there was no significant difference in ZEN+3 Se group (P>0.05). Compared with ZEN group, the AKP activity of ZEN+1.5 Se and ZEN+3 Se groups was significantly reduced (P<0.05). Western blotting results showed that compared with control group, the protein expression levels of ZO-1 and Occludin in ZEN group were significantly decreased (P<0.05). When compared with ZEN group, there were no significant differences in ZO-1 expression levels in ZEN+0.5 Se, ZEN+1.5 Se and ZEN+3 Se groups (P>0.05), but with the increase of Se concentration, the protein expression level of ZO-1 was gradually increased, the protein expression level of Occludin was significantly increased in ZEN+0.5 Se, ZEN+1.5 Se and ZEN+3 Se groups (P<0.05). The immunofluorescence results showed that selenium-oligochitosan could relieve the decreased fluorescence signal of ZO-1 and Occludin caused by ZEN, and restore the location distribution of the proteins. In conclusion, selenium-oligochitosan could reduce the increase of AKP activity in IPEC-J2 cells caused by ZEN, up-regulate the decreased expression levels of ZO-1 and Occludin in IPEC-J2 cells caused by ZEN, and adjust its location distribution.

Key words: zearalenone; selenium-oligochitosan; porcine intestinal epithelial cell (IPEC-J2); zonula occludens-1(ZO-1)

中图分类号:

S816.7

傅春妮, 李元辉, 李鹏程, 李留安, 秦顺义. 低聚壳聚糖硒抗玉米赤霉烯酮对IPEC-J2细胞紧密连接蛋白表达的影响[J]. 中国畜牧兽医, 2021, 48(11): 4311-4318.

FU Chunni, LI Yuanhui, LI Pengcheng, LI Liuan, QIN Shunyi. Effects of Selenium-oligochitosan Against Zearalenone on the Expression of Tight Junction Protein in IPEC-J2 Cells[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(11): 4311-4318.

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